ENTEROTOXIGENIC<i> BACTEROIDES FRAGILIS</i> PROMOTES CHEMORESISTANCE AND TARGETING <i>BACTEROIDES FRAGILIS</i> BY PHAGE <i>AV7</i> RESTORES CHEMOSENSITIVITY IN COLORECTAL CANCER

BACKGROUND AND AIMS: Chemoresistance is a major cause of colorectal cancer (CRC) recurrence and death. The critical role of gut microbiome in the efficacy of CRC chemotherapy remains unclear. We aim to characterize the gut microbiota signatures in responders and non-responders to chemotherapy and identify chemoresistance-associated pathogens in CRC.

METHODS: Stool samples were collected from CRC patients before chemotherapy (n=64) and analyzed by shotgun metagenomic sequencing. The effect of enterotoxigenic Bacteroides fragilis (ETBF) on chemoresistance was assessed in CRC cells and CRC mouse models of xenografts, Apc^Min/+ or azoxymethane (AOM)/dextran sodium sulfate (DSS) induced CRC tumorigenesis. Phage VA7 was used for targeting ETBF in vitro and in vivo. Bacterial adhesin-host receptor interaction was investigated by far western, biotin pull-down, and GST pull-down assays followed by mass spectrometry. RNA-seq was performed to evaluate involved pathways.
RESULTS: Bacteroides fragilis was identified as the top enriched bacterium in stool samples of CRC patients who were resistant to chemotherapy, and its abundance was associated with poor prognosis (P=0.019). Consistent with this observation, we demonstrated that ETBF co-culture with CRC cells (HT29 and HCT116) inhibited 5-FU and oxaliplatin (OXA)-induced cell apoptosis (both P<0.001). ETBF promoted chemoresistance in xenografts of nude mice (P<0.001). In particular, oral gavage of ETBF to Apc^Min/+ mice significantly attenuated the antitumor efficacy of 5-FU and OXA. Such effect was further confirmed in the second CRC tumorigenesis mouse model induced by AOM/DSS. Mechanistically, we revealed that ETBF activated Notch signaling pathway genes (Notch1, NICD, Hes1, MAML1, and c-Myc) identified by RNA-seq and confirmed by Western blot. ETBF-mediated chemoresistance critically depends on its direct interaction with CRC cells, as neither ETBF supernatant nor non-contact transwell co-culture significantly affected the efficacy of 5-FU and OXA. Concordantly, fluorescence in situ hybridization (FISH) and qPCR demonstrated the significant enrichment of ETBF in colonic tumors compared to adjacent normal tissues (P = 0.002) in mice. Scanning electron microscopy (SEM) visualized the attachment of ETBF to colon epithelial cells. ETBF surface protein SusD/RagB was identified to mediate its attachment to CRC cells. Finally, we demonstrated that ETBF-targeting phage VA7 selectively inhibited ETBF in vitro and in mice (P<0.05), and that VA7 co-administration abolished ETBF-induced chemoresistance (P<0.001).
CONCLUSION: We identified for the first time that ETBF provokes chemoresistance in CRC cells through its direct interaction with colon epithelial cells and activating Notch1 signaling. Targeting ETBF by phage VA7 is a promising therapeutic strategy to overcome ETBF-mediated chemoresistance in CRC.